RESUMO
The cyclodepsipeptide jasplakinolide (1) (aka jaspamide), isolated previously from the marine sponge Jaspis splendens, is a unique cytotoxin and molecular probe that operates through stabilization of filamentous actin (F-actin). We have recently disclosed that two analogues of 1, jasplakinolides B (3) and E, were referred to the National Cancer Institute's (NCI) Biological Evaluation Committee, and the objective of this study was to reinvestigate a Fijian collection of J. splendens in an effort to find jasplakinolide congeners with similar biological properties. The current efforts have afforded six known jasplakinolide analogues (4-7, 9, 10), two structures requiring revision (8 and 14), and four new congeners of 1 (11-13, 15) including open-chain derivatives and structures with modified ß-tyrosine residues. Compounds were evaluated for biological activity in the NCI's 60 cell line screen and in a microfilament disruption assay in both HCT-116 and HeLa cells. These two phenotypic screens provide evidence that each cytotoxic analogue, including jasplakinolide B (3), operates by modification of microfilaments. The new structure jasplakinolide V (13) has also been selected for study by the NCI's Biological Evaluation Committee. In addition, the results of a clonogenic dose-response study on jasplakinolide are presented.
Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Depsipeptídeos/farmacologia , Actinas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Citotoxinas/química , Citotoxinas/isolamento & purificação , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fiji , Células HCT116 , Células HeLa , Humanos , Biologia Marinha , Estrutura Molecular , National Cancer Institute (U.S.) , Peptídeos Cíclicos , Estereoisomerismo , Estados UnidosRESUMO
A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.
Assuntos
Produtos Biológicos , Técnicas de Química Combinatória , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Células HeLa , Humanos , Biologia Marinha , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Poríferos/química , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
A new 1-imidazoyl-3-carboxy-6-hydroxy-beta-carboline alkaloid, named hyrtiocarboline (1), was isolated from a Papua New Guinea marine sponge, Hyrtios reticulatus. The structure was elucidated from spectroscopic data, including (1)H-(15)N HMBC NMR experiments, which provided complementary (15)N chemical shift information in support of the structure. This compound showed selective antiproliferative activity against H522-T1 non-small cell lung, MDA-MB-435 melanoma, and U937 lymphoma cancer cell lines.
Assuntos
Alcaloides/isolamento & purificação , Carbolinas/isolamento & purificação , Poríferos/química , Alcaloides/química , Alcaloides/farmacologia , Animais , Carbolinas/química , Carbolinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HT29 , Células HeLa , Humanos , Masculino , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Papua Nova Guiné , EstereoisomerismoRESUMO
In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 A), while the F and G helices undergo rotations of approximately 20 degrees and center-of-mass shifts of approximately 1.4 A. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111-->Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.
Assuntos
Bacteriófago T4/genética , Metionina/química , Muramidase/química , Fragmentos de Peptídeos/química , Substituição de Aminoácidos , Fenômenos Químicos , Físico-Química , Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Muramidase/genética , Fragmentos de Peptídeos/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Selenometionina/químicaRESUMO
Bacterial biofilms are assemblages of bacterial cells and extracellular matrix that result in the creation of surface-associated macrocolony formation. Most bacteria are capable of forming biofilms under suitable conditions. Biofilm formation by pathogenic bacteria on medical implant devices has been linked to implant rejection in up to 10% of cases, due to biofilm-related secondary infections. In addition, biofilm formation has been implicated in both bacterial persistence and antibiotic resistance. In this study, a method has been developed for the discovery of small molecule inhibitors of biofilm formation in Vibrio cholerae, through the use of high-throughput epifluorescence microscopy imaging. Adaptation of a strategy for the growth of bacterial biofilms in wellplates, and the subsequent quantification of biofilm coverage within these wells, provides the first example of an image-based 384-well format system for the evaluation of biofilm inhibition in V. cholerae. Application of this method to the high-throughput screening of small molecule libraries has lead to the discovery of 29 biofilm lead structures, many of which eliminate biofilm formation without altering bacterial cell viability.
Assuntos
Biofilmes/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/fisiologia , Antibacterianos/química , Antibacterianos/farmacologia , Microscopia de Fluorescência , Fenótipo , Bibliotecas de Moléculas PequenasRESUMO
A high-throughput (HT) agar-based halo assay is described, which allows for rapid screening of chemical libraries for bioactivity in microorganisms such as yeast and bacteria. A pattern recognition algorithm was developed to identify halo-like shapes in plate reader optical density (OD) measurements. The authors find that the total growth inhibition within a detected halo provides an accurate estimate of a compound's potency measured in terms of its EC(50). The new halo recognition method performs significantly better than an earlier method based on single-point OD readings. An assay based on the halo algorithm was used to screen a 21,120-member library of drug-like compounds in Saccharomyces cerevisiae, leading to the identification of novel bioactive scaffolds containing derivatives of varying potencies. The authors also show that the HT halo assay can be performed with the pathogenic bacterium Vibrio cholerae and that liquid culture EC(50) values and halo scores show a good correlation in this organism. These results suggest that the HT halo assay provides a rapid and inexpensive way to screen for bioactivity in multiple microorganisms.
Assuntos
Algoritmos , Anti-Infecciosos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Antifúngicos/farmacologia , Bioensaio , Meios de Cultura , Ensaios de Triagem em Larga Escala/instrumentação , Concentração Inibidora 50 , Estrutura Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Fatores de Tempo , Vibrio cholerae/efeitos dos fármacosRESUMO
The goal of this study was to isolate and study additional jasplakinolide analogues from two taxonomically distinct marine sponges including two Auletta spp. and one Jaspis splendens. This led to the isolation of jasplakinolide (1) and eleven jasplakinolide analogues (3-13) including seven new analogues (6-10, 12, and 13). Structure elucidation of the new compounds was based on a combination of 1D and 2D NMR analysis, optical rotation, circular dichroism, and preparation of Mosher's esters. Five of the new compounds are oxidized tryptophan derivatives of 1, including a unique quinazoline derivative (9). Compounds 1, 3, 5-8, and 11 were evaluated in the NCI 60 cell line screen, and all compounds were tested in a microfilament disruption assay. Jasplakinolide B (11) exhibited potent cytotoxicity (GI(50) < 1 nM vs human colorectal adenocarcinoma (HCT-116) cells) but did not exhibit microfilament-disrupting activity at 80 nM.
Assuntos
Depsipeptídeos/química , Depsipeptídeos/farmacologia , Poríferos/química , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dicroísmo Circular , Depsipeptídeos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Estereoisomerismo , Triptofano/análogos & derivados , Triptofano/química , Triptofano/isolamento & purificação , Triptofano/farmacologiaRESUMO
A new compound of mixed polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS) origin, 11- O-methylpseurotin A ( 1), was identified from a marine-derived Aspergillus fumigatus. Bioassay-guided fractionation using a yeast halo assay with wild-type and cell cycle-related mutant strains of Saccharomyces cerevisiae resulted in the isolation of 1, which selectively inhibited a Hof1 deletion strain. Techniques including 1D and 2D NMR, HRESIMS, optical rotation, J-based analysis, and biosynthetic parallels were used in the elucidation of the planar structure and absolute configuration of 1. A related known compound, pseurotin A ( 2), was also isolated and found to be inactive in the yeast screen.
Assuntos
Aspergillus/química , Proteínas Associadas aos Microtúbulos/genética , Pirrolidinonas/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Candida albicans/metabolismo , Biologia Marinha , Estrutura Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Pirrolidinonas/química , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, anti-viral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being alpha-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for a hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K(i)=17microM) and selective over reticulocyte 15-hLO-1 (K(i) 15-hLO-1/12-hLO>30).
Assuntos
Plaquetas/enzimologia , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Lipoxigenase , Inibidores de Lipoxigenase/farmacologia , Técnicas de Química Combinatória , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Isoenzimas/antagonistas & inibidores , Cinética , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/química , Estrutura Molecular , Reprodutibilidade dos Testes , Relação Estrutura-AtividadeRESUMO
A re-collection of Plakortis quasiamphiaster from Vanuatu in 2003 resulted in the isolation of three known compounds, plakinidine A (1) and amphiasterins B1 (6) and B2 (7). Also isolated was a new bis-oxygenated pyrroloacridine alkaloid, plakinidine E (8), with a unique O-substitution versus N-substitution at position C-12 in 1. The biological evaluation of the active compounds in two assays provided complementary data. Plakinidine A (1) exhibited cytotoxicity against human colon H-116 cells with an IC50 of 0.23 microg/mL, but there were no effects against the yeast Saccharomyces cerevisiae diploid homozygous deletion strain of topoisomerase I (top1Delta). By contrast, 8 was inactive against H-116 cells but was potent in the yeast halo screen.
Assuntos
Alcaloides/isolamento & purificação , Antineoplásicos/isolamento & purificação , Plakortis/química , Alcaloides/química , Alcaloides/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , VanuatuRESUMO
The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.